The long-term objective of this project is to enhance the fundamental understanding of post- transcriptional mechanisms of gene regulation. The carbon storage regulatory system, Csr, studied herein, has profound effects on biofilm formation, motility, and the expression of virulence factors in ?-proteobacteria. The proposed studies detail a plan to investigate the role of DEAD-box RNA helicases in Csr regulation. The DEAD-box RNA helicases constitute a poorly understood family of regulatory proteins, whose role in bacteria was thought to be limited to rRNA maturation during cold stress, and assisting secondary cleavage events in RNA turnover. In contrast, this study addresses regulatory roles for DEAD-box RNA helicases that a) influence the expression of a global regulatory system controlling a number of bacterial virulence factors, and b) occur at temperatures relevant for mammalian infections (37C). CsrA is an RNA-binding protein that post-transcriptionally regulates numerous important bacterial genes and systems by binding to the 5' untranslated region (5' UTR) of mRNAs, thus altering protein translation and/or RNA turnover. The non-coding RNAs CsrB and CsrC bind multiple CsrA proteins with high affinity, and thereby sequester and antagonize this protein. Transcription of these ncRNAs depends upon the BarA/UvrY two-component signal transduction system. Available data suggest that the BarA/UvrY circuitry is the central input mechanism for transmitting environmental stimuli to regulation of Csr. A transposon mutagenesis screen identified the E. coli DEAD-box RNA helicases SrmB and DeaD as strong regulators of CsrB expression. SrmB and DeaD appear to affect CsrB expression independently through the BarA/UvrY system. A DeaD deletion mutant has significantly reduced UvrY levels whereas an srmB deletion exhibits reduced phosphorylation of BarA/UvrY without affecting the levels of these proteins. The focus of this proposal is to elucidate the separate pathways by which DeaD and SrmB regulate BarA/UvrY. The specific aims of this proposal are to determine how (i) DeaD and (ii) SrmB independently regulate CsrB levels and ultimately modulate CsrA activity. We will test the hypothesis that DeaD increases UvrY protein levels by promoting uvrY translation. We will combine in vivo phosphorylation assays with epistasis experiments to further characterize the effect of SrmB on UvrY phosphorylation. Finally, we will utilize genetic screens to identify factors in this pathway. The goals of this proposal are three-fold: i) determine the mechanisms by which DEAD-box RNA helicases affect Csr activity, ii) define novel in vivo targets of a class of RNA helicases whose primary activity was thought to be rRNA processing under cold- shock, and iii) develop a model system for the study of an important class of biomolecules that have diverse functions in prokaryotes, eukaryotes, and viruses.